Bovine viral diarrhea virus (“BVDV”) currently represents a major threat to the cattle industry. First described over fifty years ago, this pathogen has been found to be both highly virulent and easily spread. Considered a primary pathogen of the bovine enteric, respiratory, reproductive, and immune systems, BVDV continues to cause significant economic losses to the cattle industry worldwide. Recent outbreaks have occurred in Canada, the United States, and throughout the world.
Classified as a member of the genus Pestivirus and Flaviviridae family, BVDV is closely related to sheep border disease virus (“BDV”), and hog cholera virus (“HCV”), both of which are serologically related pestiviruses. Entire or partial genomic sequencing of pestivirus isolates has allowed the determination that a high degree of sequence conservation is present among the pestiviruses. More recently, antigenic variants of BVDV have been identified, and BVDV strains have been divided into two distinct genotypes, type 1 and type 2, which have been further subdivided, based upon cytopathogenicity. Molecular cloning, and polymerase chain reaction (PCR) technology have determined that the general structure of BVDV consists of a capsid protein and three envelope glycoproteins. The genome of BVDV is a 12.3 kb RNA consisting of a single open reading frame (“ORF”). The BVD virus is itself a small, enveloped RNA virus with positive strand polarity. This positive strand aspect of the viral genome allows the RNA to be infectious, even in the absence of virion proteins.
BVDV is spread through a herd in a fecal-oral manner, attacking the enteric, respiratory, reproductive, and immune systems. The viral load needed to provoke symptomatic infection is correlated with the type and strain of BVD virus. In addition, BVDV has the ability to infect fetuses by crossing the placenta, often resulting in a spontaneous abortion of the fetus, and a resultant decreased fertility among infected animals. Strategies for control of BVDV range from stricter management practices in an effort to simply reduce economic loss, to elaborate testing procedures to identify infected animals that, while effective, entail an unacceptable level of cost. Failure of field vaccinations for BVDV has increased the need for a test protocol that will help identify and eliminate infected animals in a cost-effective way.
It should also be noted that BVDV, like other infectious disease agents, is associated with a wide variety of clinical manifestations, creating a very difficult diagnostic challenge. Common manifestations of BVDV infection can include: abortion storms, infertility, irregular heat cycles, early embryonic deaths, fetal mummification, immuno-suppression, dysentery, thrombocytopenia, and cerebral hypoplasia. Moreover, serological studies have shown that a high percentage of cattle infected with BVDV, including those considered to be persistently infected (PI), remain clinically asymptomatic. Such conditions make it imperative that a reliable, inexpensive, and easy-to-use test be developed to assist in the detection of BVDV-infected animals in cattle herds.
The BVD virus is typically maintained in a herd due to the presence of immuno-tolerant PI carrier animals. These PI cattle are exposed to the virus in utero, but can remain clinically asymptomatic throughout the course of their lives, continually shedding fecal matter and bodily fluids with a high concentration of virus, and thereby posing the threat of infection for other animals as long as they remain in the herd. The virus may be present in more than half of the cattle in a herd before signs of an outbreak exhibit themselves. Symptoms of the disease are usually preceded by leukopenia, and testing efforts to date have focused on identifying this effect.
Prior outbreaks of BVDV have resulted in crippling economic losses to the livestock industry. For example, in Ontario in 1993, BVDV cases increased 23% in less than one year. It should also be noted that although the historical assignment of BVDV as a pestivirus was through the species it was first found to be associated with (e.g. cattle), it is now known that pestiviruses can cross species barriers. This indicates that in areas in which wild, free-ranging ruminants (e.g., moose, buffalo, etc.) are exposed to infected cattle herds, these animals are also susceptible to infection from BVDV, or can alternatively act as a reservoir of virus capable of infecting a previously “clean” herd.
Over one hundred and fifty vaccines for BVDV have been marketed to cattle farmers over the past thirty years. These vaccines have consisted of modified live BVD virus or inactivated attenuated virus and virus particles. Recent BVDV outbreaks have occurred, despite the availability and use of these vaccines. Current approaches to vaccination involve repeated yearly inoculation with vaccine for cattle, and additional steps are generally taken in an attempt to insure that no calves are born as PI carriers. However, for effective control of the BVD virus to be possible, it is essential to identify the PI animals and remove them from the herd. Several different test methods have been developed for the detection of BVDV, and/or the detection of BVDV-infected animals. These test methods include: reverse transcription-polymerase chain reaction, enzyme-linked immunoassay (ELISA), standard virus isolation techniques, and immunohistochemistry (Haines et al., “Monoclonal Antibody-Based Immunohistochemical Detection of Bovine Viral Diarrhea Virus in Formalin-Fixed, Paraffin-Embedded Tissues,” Vet. Pathol., 29:27-32 (1992)).
Both PCR and virus isolation techniques, owing to their inherent sensitivity, are each capable of detecting very low levels of BVDV. However, these methods are also time-consuming, relatively complex, and expensive. Immunohistochemistry on tissue samples, such as ear notch biopsy samples, is an effective technique for detecting PI animals. This technique, however, is time consuming, labor intensive, and requires highly trained technicians. ELISA technology, although somewhat less sensitive, is better suited as a broad-based diagnostic tool for detecting BVDV infection in animals, because it is cost effective, yields results in a short period of time, and does not require highly trained technicians and a highly specialized laboratory facility. However, antigen-capture ELISA tests for BVDV have historically relied on the use of white blood cell extracts from the animal to be tested. White blood cell extracts have been necessary because BVDV proteins accumulate to relatively high concentrations within the white blood cells of infected animals, and the previous ELISA methods lacked the sensitivity to detect their target BVDV proteins in blood serum (Horner et al., “Comparison of an Antigen Capture Enzyme-Linked Assay with Reverse Transcription-Polymerase Chain Reaction and Cell Culture Immunoperoxidase Tests for the Diagnosis of Ruminant Pestivirus Infections,” Vet. Microbiol., 43:75-84 (1995)). The preparation of white cell extracts is itself time consuming and relatively expensive, making any ELISA test reliant upon this extraction costly in and of itself.
Because the above-mentioned methods for detecting BVDV infection in animals are not only time-consuming but often require sophisticated laboratory facilities and highly trained technicians to complete, they are economically prohibitive to use in the broad fashion that is required for today's cattle industry.
More recently, a faster and more cost-effective ELISA test which detects BVDV from serum, plasma, milk, urine, or mucosal fluid samples by using a monoclonal antibody specific for BVDV viral proteins or protein fragments has been developed. See U.S. Pat. No. 6,174,667 and WO 99/15900 to Huchzermeier et al. While this method represents an advance over previously available methods, the serum samples or other bodily fluids may be difficult to collect and/or handle.
The present invention is directed to overcoming the above deficiencies in the art.